The long-term goal of this research is, through the use of cellular and animal models of cerebral ischemia, to explore novel molecular targets for therapeutic interventions in stroke. Ischemic brain injury may involve an active type of cell death reminiscent of apoptosis, especially when the ischemic insult is relatively mild and cellular energy metabolism is not irreversibly compromised. Current studies have identified a group of terminal caspases, particularly caspase-3/-7, as the central executive molecules in apoptotic ischemic neuronal death. Upon activation, caspase-3 cleaves various death substrates in the cytosol and nucleus, leading to cell death. The precise mechanism by which caspase-3 is activated in ischemic neurons is poorly understood, however, it may involve both intrinsic (cytochrome c/Apaf-1 dependent) and extrinsic (cell membrane receptor mediated) pathways. Therefore, elucidation of these signaling pathways in ischemic injury may provide novel targets for therapeutic interventions to prevent caspase-3 activation and neuronal death after stroke. Under many experimental conditions, blockage of caspase activities is able to delay, but not prevent completely, neuronal death, suggesting that a caspase-independent death pathway must be involved. A novel pro-apoptotic molecule, designated as AIF (apoptosis-inducing factor), has now been identified. AIF, which is activated and released from the mitochondria upon receiving cell death signals, potently promotes nuclear apoptosis, independent of any caspase activities. Our preliminary studies strongly suggest that AIF may function independently and synergistically with caspase-3 in the final execution of neuronal ischemic apoptosis. Therefore, the overall hypothesis underlying this proposal is that ischemia-induced neuronal cell death is mediated by both caspase-dependent and caspase-independent mechanisms, particularly the AIF-dependent pathway. The two pathways work synergistically in mediating neuronal cell death. The scientific objective of this project is to elucidate the upstream pathways for the activation of caspase-3 and AIF and to determine whether targeting these apoptosis-signaling pathways can ameliorate brain injury after transient cerebral ischemia. We propose the following specific aims: 1) Test the hypothesis that the caspase-9/Apaf-1 intrinsic pathway plays a central role in mediating caspase-3 activation and neuronal death in the hippocampus after transient forebrain ischemia; 2) Test the hypothesis that AIF and caspase-3 contribute independently and synergistically to hippocampal neuronal death after transient forebrain ischemia; 3) Test the hypotheses that AIF mediates neuronal death in cell culture models of neuronal ischemia and that Bax triggers both AIF and caspase-9/Apaf-1 pathways in ischemic neuronal death. These studies will take advantage of our recent cloning of novel rat genes encoding the dominant-negative inhibitors for caspase-9, Apaf-1, and AIF. Molecular interventions targeting the speculative cell death pathways in ischemic neurons will be achieved using the state-of-the-art TAT-fusion protein transduction technology and AAV vector-mediated neuronal gene infection.